Abstract

A major limitation of cell therapies is the rapid decline in viability
and function of transplanted cells. Here we describe a strategy to enhance cell
therapy via the conjugation of adjuvant drug-loaded nanoparticles to the
surfaces of therapeutic cells. Using this method to provide sustained
pseudo-autocrine stimulation to donor cells, we elicited dramatic enhancements
in tumor elimination in a model of adoptive T-cell therapy for cancer and
increased the in vivo repopulation rate of hematopoietic stem
cell grafts, using very low doses of adjuvant drugs that were ineffective when
given systemically. This approach is a facile and generalizable strategy to
augment cytoreagents while minimizing systemic side effects of adjuvant drugs.
In addition, these results suggest therapeutic cells are promising vectors for
actively targeted drug delivery.

Cell-based therapies, such as hematopoietic stem cell (HSC), islet cell, or
hepatocyte transplants are in routine clinical practice1,2, while new treatment strategies
implementing adult, embryonic, or induced pluripotent stem cells are in various stages
of development3,4. In the field of cancer immunotherapy, early clinical trials infusing
ex vivo-expanded tumor-specific T-lymphocytes have yielded
promising results for the treatment of cancer and chronic infections5-7. Notably, following
cell transfer, therapeutic cells often rely on the co-delivery of adjuvant drugs. These
agents are designed to maximize donor cell efficacy and in vivo
persistence, offset suppressive molecules at cell homing sites, or promote the
differentiation of transferred cells into a therapeutically optimal phenotype. Examples
include γc receptor cytokines5,8 or TGF-β signaling
inhibitors9 in adoptive T-cell therapy, or the
use of small-molecule drugs to boost immune reconstitution following HSC
transplants10. However, these agents often
require high and sustained systemic levels for efficacy. This leads to dose-limiting
toxicities for these drugs due to their generally pleiotropic activity, which has
restricted their clinical use11,12. One approach to focus adjuvant drug action on the
transferred cells is to genetically engineer donor cells to secrete their own supporting
factors13. However, regulatory and cost
barriers of large-scale clinical grade vector production and safety testing, costly and
lengthy cell culture, and technical challenges of efficient gene transfer hinder the
implementation of clinical gene therapy protocols. More importantly, several emerging
adjuvant therapies are based on small-molecule drugs that cannot be genetically
encoded9,10. Here we describe an alternate strategy for adjuvant drug delivery in
cell therapies, based on chemical conjugation of submicron-sized drug-loaded synthetic
particles directly onto the plasma membrane of donor cells, enabling continuous
pseudo-autocrine stimulation of transferred cells in vivo.

Results

Stable nanoparticle (NP) attachment to cell surfaces

To stably couple synthetic drug carrier NPs to the surface of therapeutic
cells, we exploited the fact that many cells exhibit high levels of reduced
thiol groups on their surfaces14.
Confirming prior reports, we detected substantial levels of free thiols on the
surfaces of T-cells, B-cells, and HSCs, but low amounts on red blood cells
(Fig. 1a). To link synthetic drug
carriers to cells using these surface thiols, we utilized liposomes and
liposome-like synthetic NPs 100-300 nm in diameter with a drug-loaded core and
phospholipid surface layer, where the lipid bilayer surface of the particles
included thiol-reactive maleimide headgroups (Supplementary Fig. 1). We achieved
particle conjugation by a simple two-step process (Fig. 1b): donor cells were first incubated with NPs to permit
maleimide-thiol coupling, followed by in situ PEGylation with
thiol-terminated poly(ethylene glycol) (PEG) to quench residual reactive groups
of the particles (Supplementary Fig. 2). With this approach, we could covalently link
a substantial number of NPs with diameters in the 100-300 nm range to cell types
used commonly in cell therapy, including CD8+ T lymphocytes
or lineage-Sca-1+c-kit+ HSCs
(Fig. 1c, left panels). Particles
ranging from simple liposomes (with an aqueous drug-loaded core), to more
complex multilamellar lipid NPs or lipid-coated polymer NPs15 (Fig. 1c, and
Supplementary Figs. 1 and
3) were stably attached to live cells. Importantly, particle coupling
was benign; coupling of up to 140 (±30) ~200 nm-diameter
multilamellar lipid NPs to the surface of cells was nontoxic (Supplementary Fig. 4), and blocked
only 17.2% (± 8.7%) of the total available cell surface
thiol groups (Supplementary
Fig. 5). These findings are consistent with a simple calculation of
the surface area occupied by the NPs: attachment of 150 particles each 200 nm in
diameter would occlude only 3% of the surface of a typical 7
μm-diameter T-cell. Although liposomes and lipid-coated polymer
particles spontaneously adsorbed to cell surfaces, we found that
physically-adsorbed particles were removed during mild cell washing steps, while
maleimide-linked particles remained stably bound to cells (Fig. 1d). Attachment of NPs to T-cells did not trigger
spontaneous activation of the cells (Supplementary Fig. 6), and
strikingly, particles bound to lymphocytes or HSCs remained localized at the
cell surface as revealed by optical sectioning with confocal microscopy (Fig. 1c, and Supplementary Movies 1 and 2), and
by flow cytometry internalization assays (Fig.
1e), even following extended in vitro stimulation
(Fig. 1c, right panels). In contrast,
we observed that phagocytic cells such as immature dendritic cells efficiently
internalized maleimide-functionalized NPs after a short incubation (Fig. 1e). Although all three types of NPs
tested here conjugated to lymphocytes with comparable efficiency, we chose to
focus on ~300 nm-diameter multilamellar lipid NPs (Supplementary Fig. 1b) for our
subsequent in vitro functional and in vivo
therapeutic studies, based on their high drug encapsulation efficiencies,
week-long drug release profiles, and the lack of inflammatory responses elicited
from innate immune cells exposed to the “empty” particles (Supplementary Figs. 7 and
8).

NP conjugation does not compromise key cellular functions

We next determined the maximal number of particles (without encapsulated
drug cargo) that could be linked to cells without compromising key cellular
functions, focusing on therapeutic cytotoxic T-cells that must be capable of
forming an immunological synapse and killing target cells, proliferating, and
secreting cytokines as part of their normal function. TCR-transgenic OT-1
CD8+ T-cells conjugated with up to 100 (±20) NPs
per cell retained an unmodified proliferative response after co-culture with
ovalbumin-pulsed dendritic cells; higher surface densities of particles began to
inhibit T-cell proliferation (Fig. 2a, and
Supplementary Fig.
9a,b). During cell division, surface-attached NPs segregated equally
to daughter cells, reflected by a stepwise decrease in the mean fluorescence
from cell-conjugated NPs with increasing number of divisions (Figs. 1c and ​and2a).
Attachment2a).
Attachment of at least ~100 particles/cell also did not impact T-cell
recognition/killing of ovalbumin peptide-pulsed target cells or cytokine release
profiles (Fig. 2b, and Supplementary Figure 9c). We next
assessed the impact of cell surface-tethered NPs on T-cell transmigration across
endothelial monolayers – a key capability of any therapeutic cell to
efficiently infiltrate its target tissue. We first utilized an in
vitro transwell co-culture system and quantified the migration of
NP-conjugated T-lymphocytes across a membrane-supported confluent endothelial
monolayer in response to a chemoattractant placed in the lower chamber. T-cells
carrying 100 NPs/cell exhibited unaltered transmigration efficiencies compared
to unmodified cells (Fig. 2c). After
crossing the endothelial barrier, T-cells retained 83%
(±3%) of their original NP cargo physically attached (Fig. 2d). (In comparative experiments,
liposomes and lipid-coated PLGA particles could also be carried through
endothelial layers by T-cells, though PLGA particles were not retained as well
by transmigrating cells and showed a tendency to inhibit T-cell transmigration
at high particle/cell loadings, Supplementary Fig. 10)

To determine whether in vivo tissue homing of T-cells
was affected by NP conjugation, we evaluated the tumor-homing properties of
particle-conjugated lymphocytes. Subcutaneous EL4 tumors expressing
membrane-bound Gaussia luciferase (extG-luc) and ovalbumin (EG7-OVA) or exG-luc
alone were established on opposite flanks of C57Bl/6 mice. Tumor-bearing mice
then received adoptive transfers of Firefly luciferase (F-luc)-transgenic OT-1
T-cells with or without surface-conjugated red-fluorescent NPs, or an i.v.
injection of an equivalent dose of fluorescent particles alone.
Particle-carrying OT-1 T-cells specifically trafficked to EL4-OVA tumors (Fig. 3a), and no difference in the tumor
homing potential of particle-conjugated compared to unmodified OT-1 T-cells was
observed (Fig. 3b, upper panel).
Quantitative fluorescent particle imaging of EG7-OVA tumors demonstrated that
NPs accumulated a mean 176-fold more efficiently at the tumor site when
surface-attached to OT-1 T-cells compared to systemically infused free NPs,
which were rapidly scavenged by the liver and the spleen (Fig. 3b,d). Flow cytometry analysis independently
verified that T-cell infiltration of EG7-OVA tumors was quantitatively identical
for particle-decorated and control OT-1 cells, and that the majority of
particle-conjugated cells recovered from tumors still retained their NP cargo
(Fig. 3a). In separate experiments
using fluorescently-labeled OT-1 T-cells, we confirmed prominent infiltration of
NP-decorated T-cells into EG7-OVA tumors in histological tumor sections examined
by confocal microscopy, and NPs appeared surface-localized as observed
in vitro (Fig. 3c,
Supplementary Fig. 11,
Supplementary Movie 3,4). Of note, the ability of lymphocytes to
efficiently transfer surface-tethered NPs across endothelial barriers in
vivo was not restricted to the abnormal endothelial lining16 found in tumor vasculature. When we
linked particles to resting CCR7+CD62L+
B-cells (Supplementary Fig.
12) or central memory CD8+ T-cells (data not
shown), particles were transported across the intercellular boundaries of high
endothelial venules into lymph nodes – a poorly accessible compartment
for systemically infused free NPs.

Enhanced HSC reconstitution via cell-bound adjuvant NPs

Prompted by the substantial therapeutic benefits achieved with
conjugation of cytokine-loaded particles to tumor-specific T-cells, we further
examined the utility of this new adjuvant delivery approach in the context of
hematopoietic stem cell transplantations. We chose the glycogen synthase
kinase-3 β (GSK-3β) inhibitor TWS11920 as therapeutic cargo, based on reports that
repeated high-dose bolus therapy of transplant recipients with glycogen synthase
kinase-3 (GSK-3) inhibitors enhances the repopulation kinetics of donor
HSCs10. Multilamellar lipid
nanoparticles efficiently encapsulated this small-molecule drug, and slowly
released it over a seven-day time window (Supplementary Fig. 13). We
evaluated the in vivo repopulation capabilities of
hematopoietic grafts supported by cell-bound TWS119-loaded NPs based on the
whole body photon emission from Firefly luciferase-transgenic donor HSCs, and in
separate experiments, by tracing the frequencies of GFP+
donor HSCs by flow cytometry. Following transplantation of
lineage-Sca-1+c-kit+ HSCs
from luciferase-transgenic donors into lethally-irradiated syngeneic recipients,
a steady increase in whole body bioluminescent emission was observed originating
from discrete foci over anatomic sites corresponding to the femurs, humeri,
sternum and the spleen (Fig. 5a). Whereas a
systemic TWS119 bolus injection (1.6 ng) at the time of transplantation did not
significantly alter measured engraftment kinetics (Fig. 5a,b), the same TWS119 dose encapsulated in NPs
surface-tethered to donor HSCs markedly enhanced reconstitution by HSC grafts
(median 5.7-fold higher bioluminescence than systemic TWS119 after one week,
P < 0.0001, Fig.
5a–c). Notably, animals in all treatment groups initially
engrafted HSCs in both femurs and the sternum, indicating that NP conjugation
did not compromise the intrinsic homing properties of donor HSCs. While
increasing the rate of initial reconstitution, conjugating TWS119 NPs onto HSCs
did not affect their multilineage differentiation potential, reflected by a
similar frequency of donor-derived GFP+ reconstituted cell
types compared to control HSC grafts three months after transplantation (Fig. 5d). Thus, this simple approach for
donor cell modification just prior to cell transfer can also augment
hematopoietic stem cell transplants, a procedure in routine clinical
practice.

Discussion

Cell therapies are in common clinical practice for certain indications (e.g.,
HSC and islet cell transplants) and are also being aggressively developed in other
areas of medicine, such as adoptive T-cell therapy of cancer5-7. However, many
cell therapy protocols rely on adjuvant drugs that act directly on the transferred
therapeutic cells to maintain their function, phenotype, and/or lifespan. A
ubiquitous challenge is the pleiotropic activity of many biological and
small-molecule drugs, leading to toxicity or unwanted side effects following
systemic exposure. This problem is illustrated by the use of interleukin-2 in the
support of adoptive T-cell therapy of melanoma, where IL-2 provides important
adjuvant signals to donor T-cells, but also elicits severe dose-limiting
toxicity12.

Here, we devised a facile and generalizable strategy to robustly augment the
therapeutic potential of cytoreagents, while limiting the potential for side effects
from adjuvant drugs. We showed that adjuvant agent-releasing particles can be stably
conjugated to cells without toxicity or interference with intrinsic cell functions,
follow the characteristic in vivo migration patterns of their
cellular vehicles and, ultimately, endow their carrier cells with substantially
enhanced function using low drug doses that have no effect when given by traditional
systemic routes. Prolonged retention of the particles on the surfaces of donor cells
as shown here enables sustained drug release without concerns of premature
degradation of the particle carrier or cargo due to internalization into degradative
intracellular compartments. Notably, prior work has shown that particles
~200 nm in diameter coated with anti-CD3 are readily internalized by T-cell
lines21, suggesting that internalization
of particles in the size range studied here is not impossible for lymphocytes
per se, but rather that internalization may be tightly
regulated at the cell surface– elucidating the mechanism(s) for prolonged
particle retention on T-cell and HSC surfaces is an area for future study. Numerous
reports have illustrated the potential of systemically-infused nanoparticle
materials slowly releasing drug cargos to enhance the efficacy of therapeutic drugs,
and this has led to the development of clinical products such as
anthracycline-loaded liposomes for cancer therapy22. However, in the context of support for cell therapy, our data
demonstrate that conjugation of drug-loaded particles directly to the donor cells
increases their therapeutic impact significantly (here, ~10-fold increases
in peak T-cell expansion in an adoptive T-cell therapy model for particles attached
to cells vs. the same particles systemically infused). This strategy does not
require cell preconditioning and complements traditional genetic engineering or
chemical biology approaches23 to augment or
reprogram cell function. Based on the wealth of available nanoparticle formulations
tailored to deliver small molecule drugs, proteins, siRNA, or magnetic imaging
agents24-27, the range of therapeutic or diagnostic cargos that can be attached
to therapeutic cells likely extends far beyond the small molecules and recombinant
proteins illustrated here.

Our study further demonstrates the concept of cells as chaperones that
actively direct drug-loaded nanoparticles into poorly accessible anatomical
compartments. In the field of cancer therapy, targeting strategies functionalizing
drugs or biomaterials with specific tumor-targeting ligands, such as antibodies,
aptamers, small molecules or folic acid have been demonstrated to improve
therapeutic efficacy28-30. However, these approaches generally rely on the
initially passive accumulation of targeted therapeutics at tumor sites via the
enhanced permeation and retention effect27,
and it has been shown in some systems that targeting ligands do not change the
overall tissue biodistribution of i.v.-delivered nanoparticle drug carriers, but
rather enable those particles that do reach tumors to be more efficiently
internalized by target cells28,31. In contrast, cellular nanoparticle vectors
actively transmigrate the endothelial barrier and accumulate cell-attached cargo in
tissues at >100-fold greater levels than systemically infused free
particles. This profoundly altered biodistribution opens new venues, beyond existing
cell therapies, for applications of cell products as actively targeting drug
delivery “pharmacytes” or vaccine delivery tools.

Methods

Cell lines

The murine melanoma cell line B16F10, the pancreatic islet endothelial
cell line MS1, the thymoma cell line EL4 and EG7-OVA, an EL4 cell line stably
transfected with the plasmid pAc-neo-OVA which carries a complete copy of
chicken ovalbumin (OVA) mRNA, were all purchased from the American Type Culture
Collection (ATCC). We purchased the Phoenix™ Eco retroviral packaging
cell line from Orbigen. For bioluminescent in vivo tumor imaging we retrovirally
transduced the B16F10, EL4 and EG7-OVA cell lines with a membrane-anchored form
of the Gaussia luciferase (extG-Luc), provided to us by M.
Sadelain (Memorial Sloan-Kettering Cancer Center), as described in the Supplementary
Methods.

Mice and in vivo tumor models

Animals were housed in the MIT Animal Facility. We performed all mouse
studies in the context of an animal protocol approved by the MIT Division of
Comparative Medicine following federal, state, and local guidelines. C57Bl/6
mice, C57Bl/6-Pmel-1-Thy1.1 mice, OT-1 OVA-TCR transgenic mice, and
C57Bl/6-GFP-transgenic mice were all obtained from Jackson Laboratories. C57Bl/6
(H-2Kb, Thy-1.1) firefly luciferase (F-luc)-transgenic mice 32 were provided to us by M. van den Brink
(Memorial Sloan-Kettering Cancer Center). For adoptive T-cell experiments with
OVA-specific transgenic T cells, we subcutaneously injected C57Bl/6 mice with 4
× 106 EG7-OVA tumor cells into the right flank and 2
× 106 control EL4 cells into the left flank to generate
equally sized s.c. tumors seven days later. We retrovirally transduced both
tumor cell lines with extG-luc for bioluminescent imaging. To establish melanoma
lung tumor metastases, we injected 1 × 106 B16F10-extG-luc
tumor cells i.v. via the tail vein into C57Bl/6 mice one week before T cell
treatment. On the day of adoptive Pmel-1 T cell transfer, we sublethally
irradiated recipient mice with 500 cGy of total body irradiation from a
137Cs source. All mice were treated with a single infusion of 15
× 106 effector CD8+ T cells.

Preparation of primed T-cells for adoptive transfer and retroviral
transduction

Supplementary Material

1

2

Acknowledgments

This work was supported in part by the National Institute of Health (CA140476), the
National Science Foundation (MRSEC award DMR-02-13282), Cancer Center Support (core)
grant P30-CA14051 from the National Cancer Institute, and a gift to the Koch
Institute by Curtis and Cathy Marble. DJI is an investigator of the Howard Hughes
Medical Institute.